Tagging of Tau was tested with several tags (PS-CFP2, Dendra2, CFP, and HA) and without tags, without noticeable differences in cellular distribution (supplemental Fig. to the AIS) was overexpressed. Using superresolution nanoscopy and live-cell imaging, we observed that microtubules within the AIS appeared highly dynamic, a feature essential for the TDB. Pathomechanistically, amyloid- insult caused cofilin Nitrofurantoin activation and F-actin remodeling and decreased microtubule dynamics in the AIS. Concomitantly with these amyloid-Cinduced disruptions, the AIS/TDB sorting function failed, causing AD-like Tau missorting. In summary, we provide evidence that this human and rodent Tau isoforms differ in axodendritic sorting and amyloid-Cinduced missorting and that the axodendritic distribution of Tau depends on AIS integrity. for details) (3, 4). In adult humans, the two 2N isoforms (2N3R and 2N4R) are underrepresented, only 3C4% each, compared with 16.7% each if all six isoforms were equally expressed. In adult mice, there is no 3R isoform, but the three 4R isoforms are more evenly represented (varying between 20 and 40% each). During development, the isoform ratio of human Tau shifts from predominantly 3R-Tau isoforms to a roughly even ETV4 ratio of 3R and 4R (Table 1) (4,C7). Tau is generally considered an axonal protein in mature neurons (8), but in Alzheimer disease (AD) and other tauopathies, missorting of Tau protein into the somatodendritic compartment is an early pathological event (9, 10). In cell culture and mouse models of AD and frontotemporal lobar degeneration with tauopathy (FTLD-Tau), missorting of Tau coincides with synapse loss and functional impairments (11,C13). Table 1 mRNA and protein levels of the different isoforms of Tau in human and mouse brain (5)Note that the values in column 4 do not add up to 100%, probably due to sample variance in Ref. 5. NA, not applicable. Open in a separate window Physique 1. The second repeat and inserts synergistically prevent Tau from retrograde passage through the TDB in the AIS, whereas mutations cause retrograde leakage into the soma. and and and and and in and are a graphical representation of Nitrofurantoin 2N4R-TauD2 unable to cross the diffusion barrier within the AIS (Dendra2 low and 8-repeat Tau high), but not in case of FTLD-related Tau Nitrofurantoin mutations. Note that the half-life of 2N4R-Tau is usually roughly intermediate between those of 8-repeat Tau and Kas Dendra2 alone (?), 8-repeat Tau (?), 2N4R-Tau (), and K< 0.05. ankyrin G and IV spectrin), which covers MT bundles (32). The AIS and neuronal polarity are established early in development and can be mirrored for any diagram). Transfected species of TauD2 were distributed throughout the cell (Fig. 1and of unconverted TauD2). Then 15 m of the proximal axon (50 m away from the cell body) were irradiated with UV light, causing the Dendra2 to convert from green to reddish fluorescence. This was then imaged for up to 1 h, and signals were measured in the axon or the cell body (indicated by and and half-times Dendra2 < 4K(DIV)), Tau is usually in the beginning present in all compartments. Subsequently, we photoconverted a subset of molecules in the axon to observe their propagation. As an alternative approach, to confirm the functioning of the TDB at low Tau levels, we used a protocol that limits Tau expression to 1C3-fold of endogenous Tau (estimated at 1 m (42)), which required antibody-mediated amplification of the transfected constructs. We also waited longer (4C6 days) to allow the transfected versions of Tau to reach their destiny compartment. We also used more mature well-polarized neurons (13 DIV) with longer processes from TauKO and wild-type mice. We found Nitrofurantoin that the shorter human 4R isoforms (0N4R and 1N4R) and the 3R isoforms were highly enriched in axons, whereas the longest isoform (2N4R) was in part excluded from axons (Fig. 2 (and and and (and offered relative to a Nitrofurantoin volume marker with unbiased distribution in and and and < 0.05. dendritic presence of transfected Tau (observe Materials and methods). Quantitatively, axonal e.f. was 75, 85, and 95 for 2N4R, 1N4R, and 0N4R, respectively. The 3R versions of Tau without or two inserts (0N3R and 2N3R) showed axonal enrichment but to different extents (e.f. of 90 and 95), whereas the 1N3R isoform was, with an e.f. of 70, less efficiently sorted (supplemental Table S1 and Fig. 2and anterograde penetration of the TDB), in contrast to other mutations (K280, K280PP, A152T, and all 2N4R-TauD2), which showed only slight retrograde penetration of the.